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Cellular immunological responses of pheasant during endogenous development of Eimeria colchici
Parasitology International 49 Ž2000. 147᎐154
Cellular immunological responses of pheasant during endogenous development of Eimeria colchici M. Goldova ´U , J. Pistl, V. Letkova, ´ G. Csizsmarova, ´ ´ V. Revajova, ´ A. Looszova, ´ ´ M. Levkut Uni¨ ersity of Veterinary Medicine, Komensky ´ 73, 041 81 Kosice, ˇ Slo¨ ak Republic Received 5 November 1999; received in revised form 10 March 2000; accepted 28 March 2000
Abstract We examined the time course and histological localisation of the developmental stages of Eimeria colchici. The prepatent period in the caeca of pheasants was 6 days. The patent period began on day 7 post-infection Žp.i.. and ended on day 11 p.i. with peak production of oocysts on days 8᎐9. The peripheral blood lymphocytes of pheasant chicks showed a significant increase in proliferation to E. colchici antigen from day 5 p.i., with peak on day 14 p.i.. The metabolic activity Žrespiratory burst. of heterophils increased on days 3, 4 and 14 p.i. The total number of peripheral blood leukocytes and lymphocytes in the infected pheasant chicks had increased by day 2 p.i. and reached a maximum on day 4 of the experiment. Days 5 and 6 p.i. were characterised by a drop in the number of these cells. 䊚 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Pheasant; Eimeria colchici; Schizont; Metabolic activity of phagocytes; Lymphoproliferation
1. Introduction Coccidiosis remains a major economically important disease for the poultry industry including intensively reared pheasants Ž Phasianus colchi-
U
Corresponding author. Tel.: q421-95-6229924; fax: q42195-6331816. E-mail address: [email protected] ŽM. Goldova ´..
cus .. The risk of clinical outbreaks is directly proportional to the bird population concentration. The first reference to coccidiosis in pheasants was made by Tyzzer w1x. A review of seven Eimeria species in pheasants was published by Pellerdy ´ w2x. Norton w3x summarised the description of 10 Eimeria species affecting intensively reared pheasant chicks. According to Norton w3x only three species ᎏ Eimeria colchici, E. duodenalis
1383-5769r00r$ - see front matter 䊚 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 3 8 3 - 5 7 6 9 Ž 0 0 . 0 0 0 4 3 - X
148
M. Goldo¨´ a et al. r Parasitology International 49 (2000) 147᎐154
and E. phasiani ᎏ are associated with coccidiosis w4x reported that E. colchici in the UK. Bejsovec ˇ was the species most commonly encountered in pheasants in the Czech Republic. Current coccidiosis-control strategies depend heavily upon prophylactic feeding of anti-coccidial drugs. This approach to coccidiosis control is running into controversy as consumer pressure for drug-free poultry is increasing. New approaches based upon genetics and naturally evoked immunity are the current targets of coccidiosis research w5x. For further progress in developing anti-coccidial treatments, a clear understanding of coccidial pathogenesis and the specific mechanisms involved in the effective protective responses normally induced by infection are necessary. These responses, which are operative within the submucosa of the intestinal wall, include both antibody production and the development of an array of cell-mediated responses, the latter being most important for protection w6᎐8x. The cellular response in the gut is a prominent feature of coccidiosis w9,10x. Attempts have been made to correlate it with immunity w11x. Host responses to intracellular parasites involve many facets of the immune response, including non-specific as well as antigen-specific components, however, there is no convincing evidence for any significant role of antibodies in anticoccidial immunity w12,13x. The metabolic activity Žrespiratory burst. assessment of the heterophils in our study has been chosen from the non-specific peripheral blood immune response indicators, because information on the respiratory burst in chicken heterophils is limited w14x. Heterophils, the avian equivalents to the mammalian neutrophil, are highly phagocytic, polymorphonuclear white blood cells which are important mediators of innate resistance in poultry; especially in young birds that have not yet developed an acquired immune response w15x. On the other hand, sporozoites, merozoites and soluble proteins secreted from cultured parasites stimulate T-cell proliferation and in vitro lymphoproliferation is a very useful assay for evaluation of antigen-specific immune response w16,17x. Our current work explores the endogenous developmental phase of E. colchici in the caeca of
pheasant chicks and its influence on metabolic activity of the peripheral blood heterophils and in vitro antigen-specific proliferation of lymphocytes.
2. Materials and methods 2.1. Isolation of pure strains of Eimeria colchici The pure culture of E. colchici was obtained by isolating single oocysts on agar w18x. Pheasant chicks were hatched and kept in a chick breeder in sterile conditions. Fifty coccidia-free 2-week-old pheasant chicks were infected intraproventricularly with a single oocyst. Oocysts were collected from their faeces and were used for experimental infection after sporulation in 2% potassium bichromate solution. 2.2. Experimental animals and experimental design One hundred 2-week-old coccidia-free pheasant chicks were experimentally infected with a pure suspension of sporulated E. colchici oocysts Ž5000 oocysts per chick.. Blood samples for immunological tests, and intestinal tissue samples for histological examination were obtained at 24-h intervals, i.e. 24, 48, 72, 96, 120 and 144 h p.i. Additional blood samples were collected on days 14 and 21 p.i. 2.3. Histological in¨ estigations Tissue samples from caeca were fixed in 4% formalin and subjected to routine paraffin processing. Histological sections Ž8 m thick. were stained with haematoxylin-eosin and examined for histopathological changes and morphological determination of the developmental stages of E. colchici. 2.4. Isolation and purification of sporozoites A suspension of E. colchici oocysts was sterilised on their surface with sodium hypochlorite. After centrifugation Ž2500 rev. miny1 for 10 min. the supernatant was decanted and the oocysts
M. Goldo¨´ a et al. r Parasitology International 49 (2000) 147᎐154
were resuspended in 20 ml PBS pH 7.6, in a round-bottomed centrifuge tube. Glass beads Ž2 mm in diameter. were added so that they comprised approximately half the resulting volume, and the contents were agitated until most of the oocysts were mechanically fractured, thus releasing their sporocysts. The progress of the releasing process was monitored by microscopic examination at regular intervals. Sporocysts were recovered from the suspension by sieving and re-suspending in buffer. Fifty milligrams of trypsin and 100 mg of bile salts were added, and the mixture was incubated for 30 min at 41⬚C until most of the sporozoites were excysted. The sporozoites were then twice washed in PBS pH 7.6 and then filtrated through a simple column w19x. The concentration of sporozoites was adjusted to 5 = 10 6 mly1 . 2.5. Preparation of leukocytes Blood samples were collected by cardiac puncture into tubes containing sodium heparin Ž20 units mly1 of blood.. The number of leukocytes and lymphocytes in whole blood was evaluated by routine laboratory methods. Leukocytes were isolated by a method described by Ander and Latimer w20x with a slight modification by Agrawal and Reynolds w21x and Barta et al. w17x. A two-step discontinuous Ficoll-sodium diatrizoate gradient was prepared by using commercially available reagents, 3 ml of Histopaque-1077 Ždensity 1.077; Sigma Chemical Co.. was layered over 3 ml of Histopaque-1119 Ždensity 1.119; Sigma. and placed in a 15-ml disposable centrifuge tube. Two millilitres of blood diluted with the same volume of phosphate buffered saline ŽPBS. was carefully layered over the Histopaque-1077 and centrifuged at 300 = g for 40 min at room temperature. After centrifugation, the layer of lymphocytes at the plasmarHistopaque-1077 interface and heterophil-rich Histopaque-1119 layer were transferred to separate sterile plastic tubes. Both suspensions of leukocytes were separately washed three times in RPMI 1640 medium and resuspended in the same medium to a final concentration of 1 = 10 7 cells mly1 . Viability of cells in
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suspensions was determined by the trypan blue dye and was over 95%. 2.6. The iodo-nitro-tetrazolium reductase test Iodo-nitro-tetrazolium reductase test ŽINTT. was carried out for quantitative evaluation of the metabolic activity ŽMA. of phagocytes according to the method of Lokaj and Oburkova w22x. This method was used to measure the respiratory burst of heterophils in the process of in vitro phagocytosis of starch during infection of pheasant chicks with E. colchici. Suspensions of 0.2 ml of heterophiles Ž1 = 10 7 mly1 . in RPMI 1640 medium were divided into two sterile plastic tubes. One portion of cells was incubated with 0.1 ml of 1% starch suspension ŽAmylum oryzae. in PBS and the other without starch Ž0.1 ml PBS.. In addition to this, each tube contained 0.2 ml of 0.1% INT Ž3r4-iodophenyl-2-r4-nitrophenylr-5-phenylrtetrazolium chloride; Lachema Brno. and medium added to 1 ml. Each suspension was tested in triplicate. After incubation at 40⬚C for 45 min and lysis of cells by acetone, the content of red-coloured formasan, which had arisen due to the reduction of INT in heterophiles, was determined spectrophotometerically at 485 nm. The results were recorded in the form of an index of metabolic activity ŽIMA. based on the ratio of mean optical density ŽOD485 . of the suspensions of heterophiles with starch and the leucocyte suspension without starch. 2.7. The lymphoproliferation test A colorimetric immunoassay was used for the quantification of cell proliferation to determine the antigen-specific response of lymphocytes. This is based on the measurement of incorporation of 5-bromo-2⬘-deoxyuridine ŽBrdU. during DNA synthesis ŽBrdU-colorimetric cell proliferation ELISA kit, Boehringer Mannheim.. Suspensions of lymphocytes Ž100 l of 2 = 10 6 cells per well in RPMI 1640 with 10% of foetal calf serum w17x. were cultured with antigen Ž10 l of a suspension of E. colchici sporozoites at a concentration of 5 = 10 6 mly1 . and without antigen in 96-well
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microtiter test system at 37⬚C in a humid atmosphere with 5% CO 2 for 96 h. Each culture was tested in triplicate. BrdU, in resulting concentration 100 M, was added and the cells were reincubated 18 h before the end of cultivation. After the removal of the medium, fixation and DNA denaturation, 100 l anti BrdU-peroxidase labelled conjugate was added for 90 min at room temperature. The immune complexes were detected by the subsequent substrate Ž100 l of substrate solution. reaction product in a 30-min period at room temperature. Reaction was stopped by 25 l 1 M H 2 SO4 and quantitated by the absorbance, measured in an ELISA reader at 450 nm. Cell activation rate was expressed as a stimulation index ŽSI. calculated on the basis of the ratio of absorbance of the stimulated cells to the non-stimulated cells.
Fig. 2. Macro- and microgametocytes of E. colchici in the caeca 120 h p.i. Hematoxylin-eosin, 1000 = .
3. Results
2.8. Statistical analysis For statistical evaluation of the results Student’s test was used.
Fig. 1. First generation schizonts of E. colchici in the crypts of the caeca 24 h p.i. Hematoxylin-eosin, 1000 = .
Numerous first-generation schizonts were found in the posterior portion of the small intestine and in the intestinal crypts of the caeca 24 h p.i. ŽFig. 1.. Second-generation schizonts were observed 72 h p.i. The first gametocytes were recorded as early as day 4 p.i. in the caeca, and at 120 h p.i. numerous macro- and micro-gamonts could be seen, thus indicating the beginning of the sexual phase of the endogenous cycle of E. colchici Ž Fig. 2.. The patent period began on day 7 and ended on day 11 p.i. with the largest production of oocysts on days 8᎐9 ŽFig. 3..
Fig. 3. Oocyst output per pheasant chick on day 7᎐11 p.i.
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Infection with E. colchici resulted in a significant increase in the absolute number of leukocytes and lymphocytes from day 2 p.i. ŽTable 1.. The response of peripheral blood lymphocytes to E. colchici antigen in the lymphoproliferation test increased significantly from day 5 Ž P- 0.05., day 6 Ž P- 0.01., and was greatest on the last day measured Žday 14, P- 0.001; when compared to the control values; Fig. 4.. The metabolic activity of peripheral blood phagocytes increased significantly on days 3 and 4 Ž P- 0.05., and 14 p.i. when compared to the control values ŽFig. 5..
4. Discussion Hatchling poultry exhibit a transient susceptibility to infectious diseases during their first week of life due to a qualitative impairment of the avian natural and acquired host defences w23x. With Eimeria infections, hatchlings are most highly susceptible during the first 4 days, after which they become more resistant to infection. This period of ‘transient immunoincompetence’ is characterised by a general failure of the T-cells to proliferate and secrete cytokines and a functional immaturity of heterophils in the first week of life w24,23,25x. The pathogenic caecal species, E. colchici, which is the causative agent of acute coccidiosis
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in pheasants, is characterised by high morbidity and mortality rates, and produces a large number of oocysts in a white caseous exudate. According to Norton w3x the endogenous phase of E. colchici begins in the central portion of the small intestine with the formation of schizonts of the first generation 72᎐96 h p.i. The formation of second generation schizonts is described in the posterior portion of the small intestine. Norton w3x observed the infection of the caeca by the first schizonts of the third-generation as late as 96᎐120 h p.i. Oocyst production was observed 144 h after infection. In contrast, in our experiment first generation schizonts were found in the ileum and principally in the intestinal crypts of the caeca on day 1 p.i. w26x. Pellerdy ´ w2x reported that first generation schizonts released merozoites 60 h p.i. in the same portion of the intestine as in our trial. w2x found the beginning of gametogony Pellerdy ´ from the 106th h in the caeca, while the third shizogony cycle was still in progress, and the first oocysts were discharged 6 days after infection. Cell-mediated immunity plays a major role in the poultry’s resistance to coccidiosis. Complex interactions involving coccidian parasites with various sub-populations of intestinal lymphocytes are a characteristic feature of the gut immune responses to this intracellular microorganism w5x. After primary infection there were, in both the epithelium and the lamina propria, two distinct increases in the number of T-lymphocytes on days
Table 1 Absolute counts of leukocytes and lymphocytes in the peripheral blood of pheasant chicks infected by Eimeria colchici a Days after infection
1 2 3 4 5 6 14 Control 1᎐6 Control 14 a
Absolute numbers Ž1 = 109 ly1 . " S.D. Leukocytes
Lymphocytes
10.05" 2.72 19.58" 6.99U 19.77" 8.73U 20.13" 0.88UUU 13.14" 7.78 11.96" 2.46 27.55 " 5.06 10.22" 0.58 23.57" 2.85
8.58" 2.38 17.37" 5.86UU 17.12" 7.84U 18.53" 1.06UUU 11.12" 6.63 9.94" 2.24 23.43" 3.73 8.55" 1.16 21.55" 3.14
Significantly different from the days 1᎐6 controls: U P- 0.05; UU P- 0.01; UUU P- 0.001.
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Fig. 4. Proliferation of lymphocytes from the peripheral blood of pheasant chicks infected by E. colchici.
3᎐5 and then on day 11 after infection w8x. Our results demonstrate that the oral infection of pheasant chicks with coccidia results in lymphocytosis and leucocytosis at the beginning of the infection. Similar changes in the numbers of these cells have been observed in chickens and rats infected with coccidia w27x. The gradual decrease
of peripheral blood lymphocytes and leukocytes after day 4 may be due to the infiltration of lymphocytes into the intestinal mucosa at the site of infection w28,29x. The drop in the level of white blood cells after day 4 p.i. also coincides with the beginning of the sexual phase of the endogenous cycle of E. colchici in the colon of infected pheas-
Fig. 5. Index of metabolic activity of phagocytes from the peripheral blood of pheasant chicks infected by E. colchici.
M. Goldo¨´ a et al. r Parasitology International 49 (2000) 147᎐154
ants w26x. This closely coincides with the significant increase in the index of metabolic activity ŽIMA. of phagocytes, which was noted on day 3, 4 and 14 p.i. The increase of the IMA mainly in pheasant chicks of experimental group and the total number of leukocytes and lymphocytes on day 14 in both experimental and control pheasant chicks was possibly due the secondary bacterial infection of pheasant chicks in combination with age factors, because heterophil activities are agedependent in poultry with optimal levels at 14᎐21 days post-hatch with simultaneous development of cell-mediated immune responsiveness w24,23,25x. A significant increase in the proliferative activity of peripheral blood lymphocytes to E. colchici antigen on day 5 and 6 was noted with a maximum on day 14 p.i. Similar results were described by Lillehoj w30x in experimental coccidiosis of chickens. T-cell proliferative response to sporozoite antigens was detected on days 7᎐10 p.i. The peak T-cell proliferative response was observed 14 days p.i. and quickly waned after 21 day p.i. A direct relationship between resistance to infection E. tenella in chickens and lymphoproliferation of peripheral blood lymphocytes in response to parasite antigen was described w31x. In conclusion, these studies indicate that E. colchici infection of pheasant chicks resulted in a patent period lasting from day 7᎐11 p.i., with the largest production of oocysts occurring on days 8᎐9. The populations of peripheral blood heterophils and lymphocytes reflect the cellular immune response in a coccidial infection of pheasant chicks, which is characterised by leucocytosis at the beginning of coccidial infection, with increased phagocytosis of the heterophils and the proliferation response of lymphocytes to sporozoite antigens.
Acknowledgements This work was supported by a research grant from the Scientific Grant Agency of The ministry of Education of The Slovak Republic and Slovak Academy of Sciences ŽGrant 1r4254r97..
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References w1x Tyzzer EE. Coccidiosis in gallinaceous birds. Am J Hyg 1929;10:269᎐271. w2x Pellerdy LP. Coccidia and coccidiosis. 2 Berlin: Parey ´ Verlag, 1974:959. w3x Norton CC. Coccidia of the pheasant. Folia Vet Lat 1976;6:218᎐238. w4x Bejsovec J. Coccidiosis in the pheasant Phasianus ˇ colchicus L. and in the partridge Perdix perdix L. in an agricultural area of Czechoslovakia. J Protozool 1972;19:75. w5x Lillehoj HS. Immunity and host genetic-based control strategies for avian coccidiosis. Suppl World Poultry ᎏ coccidiosis special. Misset, 1996:17᎐19. w6x Wakelin D, Rose ME. Immunity to coccidiosis. In: Long PL, editor. Coccidiosis of man and domestic animals. Boca Raton, USA: CRC Press, 1990:281᎐306. w7x Lillehoj HS, Bacon LD. Increase of intestinal intraepithelial lymphocytes expressing CD8 antigen following challenge infection with Eimeria acer¨ ulina. Avian Dis 1991;35:294᎐301. w8x Rothwell L, Gramzinski RA, Rose ME, Kaiser P. Avian coccidiosis: changes in intestinal lymphocyte populations associated with the development of immunity to Eimeria maxima. Parasite Immunol 1995;17:525᎐533. w9x Barber IK. Pathological processes associated with coccidiosis. In: Barta JR, Fernando MA, editors. Proceedings of the VIth International Coccidiosis Conference University of Guelph, Canada, 1993: 81᎐94. w10x Trout JM, Lillehoj HS. Eimeria acer¨ ulina infection: evidence for the involvement of CD8q T lymphocytes in sporozoite transport and host protection. Poult Sci 1995;74Ž7.:1117᎐1125. w11x Rose ME, Millard BJ, Hesket P. Intestinal changes associated with the expression of immunity to challenge with E. ¨ ermiformis. Infect Immun 1992;60:5283᎐5292. w12x Lillehoj HS. Influence of inoculation dose, inolulation schedule, host age, and host genetics on disease susceptibility and development of Eimeria tenella infection. Avian Dis 1987;32:437᎐441. w13x Rose ME. Eimeria, Isospora and Cryptosporidium in immune response in parasitic infections. In: Soulsby EJL, editor. Immunology, immunopathology, immunoprophylaxis. Boca Raton, Fl: CRC Press, 1987:275. w14x Fitko R, Jakubowski K, Roszko E, Zielinski H, Potrzuska I. The level of corticosterone and some indices of immunity of chickens in the course of immobilization stress and after the administration of immunomodifying drugs. Med Welt 1992;48Ž7.:289᎐300. In Polish. w15x Powell PC. Immune mechanisms in infections of poultry. Vet Immunol Immunopathol 1987;15:87᎐113. w16x Lillehoj HS. Cell-mediated immunity in parasitic and bacterial diseases. In: Sharma JM, editor. Avian cellular immunology. CRC PRESS Inc, 1991:155᎐182.
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w17x Barta O, Barta V, Pierson FW. Optimum conditions for the chicken lymphocyte transformation test. Avian Dis 1992;36:945᎐955. w18x Tsutsumi Y. Eimeria tsunodai sp. nov. ŽProtozoa: Eimeriidae. a caecal coccidium of japaneses quails ŽCoturnix coturnix japonica.. Jpn J Vet Sci 1972;34:1᎐9. w19x Shirley MV. 1995. Eimeria species and strains of chickens. In: COST 89r820 Biotechnology. Guidelines on techniques in coccidiosis research. EC Luxembourg: 1᎐25. w20x Andersen CB, Latimer KS. Separation of avian heterophils from blood using Ficoll᎐Hypaque discontinuous gradients. Avian Dis 1989;33:163᎐169. w21x Agrawal PK, Reynolds D. Evaluation of the cell-mediated immune response of chickens vaccinated with Newcastle Disease Virus as determined by the under-agarose leukocyte-migration-inhibition technique. Avian Dis 1991;35:360᎐364. w22x Lokaj V, Oburkova P. Stanovenı´ tetrazolim-reduktazove ´ ´ aktivity leukocytu. ´ Imunol Zprav 1975;6:42᎐44. In Czech. w23x Lowenthal JW, Connick TE, McWaters PG, Zork JJ. Development of T cell immune responsiveness in the chicken. Immunol Cell Biol 1994;72:115᎐122. w24x Suresh M, Sharma JM, Belzer SW. Studies on lymphocyte subpopulations and effect of age on immune competence in turkeys. Dev Comp Immunol 1993;17: 525᎐535.
w25x Lowry VK, Genovese KJ, Bowden LL, Kogut MH. Ontogenic development of the phagocytic and bactericidal activities of turkey heterophils and their potentiation by Salmonella enteritidis ᎏ immune lymphokines. FEMS Immunol Microbiol 1997;19:95᎐100. w26x Goldova ´ M, Csizsmarova ´ ´ G, Letkova´ V. Course of the endogenous developmental phase of selected Eimeria species in pheasant. Vet Parasitol 1998;77:289᎐295. w27x Rose ME, Hesketh P, Ogilvie BM. Peripheral blood leukocyte response to coccidial infection: a comparison of the response in rats and chickens and its correlation with resistance to infection. Immunology 1979;36:71᎐79. w28x Goldova ´ M, Csizsmarova ´ ´ G, Letkova´ V, Kocisˇ J, Kolodzieysky L. Course of endogenous developmental phase of Eimeria colchici and Eimeria duodenalis in pheasants. Veterinarstvı ´ ´ 1993;43:286᎐287. In Slovak. w29x Lillehoj HS. Role of T lymphocytes and cytokines in coccidiosis. Int J Parasitol 1998;28:1071᎐1081. w30x Lillehoj HS. Immune response during coccidiosis in SC FP chickens. I In vitro assessment of T cell proliferation to stage-specific parasite antigens. Vet Immunol Immunopathol 1986;12:321᎐327. w31x Bumstead JM, Bumstead N, Rothwell L, Tomley FM. Comparison of immune responses in inbred lines if chickens to Eimeria maxima and Eimeria tenella. Parasitology 1995;111:143᎐151.
Cellular immunological responses of pheasant during endogenous development of Eimeria colchici M. Goldova ´U , J. Pistl, V. Letkova, ´ G. Csizsmarova, ´ ´ V. Revajova, ´ A. Looszova, ´ ´ M. Levkut Uni¨ ersity of Veterinary Medicine, Komensky ´ 73, 041 81 Kosice, ˇ Slo¨ ak Republic Received 5 November 1999; received in revised form 10 March 2000; accepted 28 March 2000
Abstract We examined the time course and histological localisation of the developmental stages of Eimeria colchici. The prepatent period in the caeca of pheasants was 6 days. The patent period began on day 7 post-infection Žp.i.. and ended on day 11 p.i. with peak production of oocysts on days 8᎐9. The peripheral blood lymphocytes of pheasant chicks showed a significant increase in proliferation to E. colchici antigen from day 5 p.i., with peak on day 14 p.i.. The metabolic activity Žrespiratory burst. of heterophils increased on days 3, 4 and 14 p.i. The total number of peripheral blood leukocytes and lymphocytes in the infected pheasant chicks had increased by day 2 p.i. and reached a maximum on day 4 of the experiment. Days 5 and 6 p.i. were characterised by a drop in the number of these cells. 䊚 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Pheasant; Eimeria colchici; Schizont; Metabolic activity of phagocytes; Lymphoproliferation
1. Introduction Coccidiosis remains a major economically important disease for the poultry industry including intensively reared pheasants Ž Phasianus colchi-
U
Corresponding author. Tel.: q421-95-6229924; fax: q42195-6331816. E-mail address: [email protected] ŽM. Goldova ´..
cus .. The risk of clinical outbreaks is directly proportional to the bird population concentration. The first reference to coccidiosis in pheasants was made by Tyzzer w1x. A review of seven Eimeria species in pheasants was published by Pellerdy ´ w2x. Norton w3x summarised the description of 10 Eimeria species affecting intensively reared pheasant chicks. According to Norton w3x only three species ᎏ Eimeria colchici, E. duodenalis
1383-5769r00r$ - see front matter 䊚 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 3 8 3 - 5 7 6 9 Ž 0 0 . 0 0 0 4 3 - X
148
M. Goldo¨´ a et al. r Parasitology International 49 (2000) 147᎐154
and E. phasiani ᎏ are associated with coccidiosis w4x reported that E. colchici in the UK. Bejsovec ˇ was the species most commonly encountered in pheasants in the Czech Republic. Current coccidiosis-control strategies depend heavily upon prophylactic feeding of anti-coccidial drugs. This approach to coccidiosis control is running into controversy as consumer pressure for drug-free poultry is increasing. New approaches based upon genetics and naturally evoked immunity are the current targets of coccidiosis research w5x. For further progress in developing anti-coccidial treatments, a clear understanding of coccidial pathogenesis and the specific mechanisms involved in the effective protective responses normally induced by infection are necessary. These responses, which are operative within the submucosa of the intestinal wall, include both antibody production and the development of an array of cell-mediated responses, the latter being most important for protection w6᎐8x. The cellular response in the gut is a prominent feature of coccidiosis w9,10x. Attempts have been made to correlate it with immunity w11x. Host responses to intracellular parasites involve many facets of the immune response, including non-specific as well as antigen-specific components, however, there is no convincing evidence for any significant role of antibodies in anticoccidial immunity w12,13x. The metabolic activity Žrespiratory burst. assessment of the heterophils in our study has been chosen from the non-specific peripheral blood immune response indicators, because information on the respiratory burst in chicken heterophils is limited w14x. Heterophils, the avian equivalents to the mammalian neutrophil, are highly phagocytic, polymorphonuclear white blood cells which are important mediators of innate resistance in poultry; especially in young birds that have not yet developed an acquired immune response w15x. On the other hand, sporozoites, merozoites and soluble proteins secreted from cultured parasites stimulate T-cell proliferation and in vitro lymphoproliferation is a very useful assay for evaluation of antigen-specific immune response w16,17x. Our current work explores the endogenous developmental phase of E. colchici in the caeca of
pheasant chicks and its influence on metabolic activity of the peripheral blood heterophils and in vitro antigen-specific proliferation of lymphocytes.
2. Materials and methods 2.1. Isolation of pure strains of Eimeria colchici The pure culture of E. colchici was obtained by isolating single oocysts on agar w18x. Pheasant chicks were hatched and kept in a chick breeder in sterile conditions. Fifty coccidia-free 2-week-old pheasant chicks were infected intraproventricularly with a single oocyst. Oocysts were collected from their faeces and were used for experimental infection after sporulation in 2% potassium bichromate solution. 2.2. Experimental animals and experimental design One hundred 2-week-old coccidia-free pheasant chicks were experimentally infected with a pure suspension of sporulated E. colchici oocysts Ž5000 oocysts per chick.. Blood samples for immunological tests, and intestinal tissue samples for histological examination were obtained at 24-h intervals, i.e. 24, 48, 72, 96, 120 and 144 h p.i. Additional blood samples were collected on days 14 and 21 p.i. 2.3. Histological in¨ estigations Tissue samples from caeca were fixed in 4% formalin and subjected to routine paraffin processing. Histological sections Ž8 m thick. were stained with haematoxylin-eosin and examined for histopathological changes and morphological determination of the developmental stages of E. colchici. 2.4. Isolation and purification of sporozoites A suspension of E. colchici oocysts was sterilised on their surface with sodium hypochlorite. After centrifugation Ž2500 rev. miny1 for 10 min. the supernatant was decanted and the oocysts
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were resuspended in 20 ml PBS pH 7.6, in a round-bottomed centrifuge tube. Glass beads Ž2 mm in diameter. were added so that they comprised approximately half the resulting volume, and the contents were agitated until most of the oocysts were mechanically fractured, thus releasing their sporocysts. The progress of the releasing process was monitored by microscopic examination at regular intervals. Sporocysts were recovered from the suspension by sieving and re-suspending in buffer. Fifty milligrams of trypsin and 100 mg of bile salts were added, and the mixture was incubated for 30 min at 41⬚C until most of the sporozoites were excysted. The sporozoites were then twice washed in PBS pH 7.6 and then filtrated through a simple column w19x. The concentration of sporozoites was adjusted to 5 = 10 6 mly1 . 2.5. Preparation of leukocytes Blood samples were collected by cardiac puncture into tubes containing sodium heparin Ž20 units mly1 of blood.. The number of leukocytes and lymphocytes in whole blood was evaluated by routine laboratory methods. Leukocytes were isolated by a method described by Ander and Latimer w20x with a slight modification by Agrawal and Reynolds w21x and Barta et al. w17x. A two-step discontinuous Ficoll-sodium diatrizoate gradient was prepared by using commercially available reagents, 3 ml of Histopaque-1077 Ždensity 1.077; Sigma Chemical Co.. was layered over 3 ml of Histopaque-1119 Ždensity 1.119; Sigma. and placed in a 15-ml disposable centrifuge tube. Two millilitres of blood diluted with the same volume of phosphate buffered saline ŽPBS. was carefully layered over the Histopaque-1077 and centrifuged at 300 = g for 40 min at room temperature. After centrifugation, the layer of lymphocytes at the plasmarHistopaque-1077 interface and heterophil-rich Histopaque-1119 layer were transferred to separate sterile plastic tubes. Both suspensions of leukocytes were separately washed three times in RPMI 1640 medium and resuspended in the same medium to a final concentration of 1 = 10 7 cells mly1 . Viability of cells in
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suspensions was determined by the trypan blue dye and was over 95%. 2.6. The iodo-nitro-tetrazolium reductase test Iodo-nitro-tetrazolium reductase test ŽINTT. was carried out for quantitative evaluation of the metabolic activity ŽMA. of phagocytes according to the method of Lokaj and Oburkova w22x. This method was used to measure the respiratory burst of heterophils in the process of in vitro phagocytosis of starch during infection of pheasant chicks with E. colchici. Suspensions of 0.2 ml of heterophiles Ž1 = 10 7 mly1 . in RPMI 1640 medium were divided into two sterile plastic tubes. One portion of cells was incubated with 0.1 ml of 1% starch suspension ŽAmylum oryzae. in PBS and the other without starch Ž0.1 ml PBS.. In addition to this, each tube contained 0.2 ml of 0.1% INT Ž3r4-iodophenyl-2-r4-nitrophenylr-5-phenylrtetrazolium chloride; Lachema Brno. and medium added to 1 ml. Each suspension was tested in triplicate. After incubation at 40⬚C for 45 min and lysis of cells by acetone, the content of red-coloured formasan, which had arisen due to the reduction of INT in heterophiles, was determined spectrophotometerically at 485 nm. The results were recorded in the form of an index of metabolic activity ŽIMA. based on the ratio of mean optical density ŽOD485 . of the suspensions of heterophiles with starch and the leucocyte suspension without starch. 2.7. The lymphoproliferation test A colorimetric immunoassay was used for the quantification of cell proliferation to determine the antigen-specific response of lymphocytes. This is based on the measurement of incorporation of 5-bromo-2⬘-deoxyuridine ŽBrdU. during DNA synthesis ŽBrdU-colorimetric cell proliferation ELISA kit, Boehringer Mannheim.. Suspensions of lymphocytes Ž100 l of 2 = 10 6 cells per well in RPMI 1640 with 10% of foetal calf serum w17x. were cultured with antigen Ž10 l of a suspension of E. colchici sporozoites at a concentration of 5 = 10 6 mly1 . and without antigen in 96-well
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microtiter test system at 37⬚C in a humid atmosphere with 5% CO 2 for 96 h. Each culture was tested in triplicate. BrdU, in resulting concentration 100 M, was added and the cells were reincubated 18 h before the end of cultivation. After the removal of the medium, fixation and DNA denaturation, 100 l anti BrdU-peroxidase labelled conjugate was added for 90 min at room temperature. The immune complexes were detected by the subsequent substrate Ž100 l of substrate solution. reaction product in a 30-min period at room temperature. Reaction was stopped by 25 l 1 M H 2 SO4 and quantitated by the absorbance, measured in an ELISA reader at 450 nm. Cell activation rate was expressed as a stimulation index ŽSI. calculated on the basis of the ratio of absorbance of the stimulated cells to the non-stimulated cells.
Fig. 2. Macro- and microgametocytes of E. colchici in the caeca 120 h p.i. Hematoxylin-eosin, 1000 = .
3. Results
2.8. Statistical analysis For statistical evaluation of the results Student’s test was used.
Fig. 1. First generation schizonts of E. colchici in the crypts of the caeca 24 h p.i. Hematoxylin-eosin, 1000 = .
Numerous first-generation schizonts were found in the posterior portion of the small intestine and in the intestinal crypts of the caeca 24 h p.i. ŽFig. 1.. Second-generation schizonts were observed 72 h p.i. The first gametocytes were recorded as early as day 4 p.i. in the caeca, and at 120 h p.i. numerous macro- and micro-gamonts could be seen, thus indicating the beginning of the sexual phase of the endogenous cycle of E. colchici Ž Fig. 2.. The patent period began on day 7 and ended on day 11 p.i. with the largest production of oocysts on days 8᎐9 ŽFig. 3..
Fig. 3. Oocyst output per pheasant chick on day 7᎐11 p.i.
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Infection with E. colchici resulted in a significant increase in the absolute number of leukocytes and lymphocytes from day 2 p.i. ŽTable 1.. The response of peripheral blood lymphocytes to E. colchici antigen in the lymphoproliferation test increased significantly from day 5 Ž P- 0.05., day 6 Ž P- 0.01., and was greatest on the last day measured Žday 14, P- 0.001; when compared to the control values; Fig. 4.. The metabolic activity of peripheral blood phagocytes increased significantly on days 3 and 4 Ž P- 0.05., and 14 p.i. when compared to the control values ŽFig. 5..
4. Discussion Hatchling poultry exhibit a transient susceptibility to infectious diseases during their first week of life due to a qualitative impairment of the avian natural and acquired host defences w23x. With Eimeria infections, hatchlings are most highly susceptible during the first 4 days, after which they become more resistant to infection. This period of ‘transient immunoincompetence’ is characterised by a general failure of the T-cells to proliferate and secrete cytokines and a functional immaturity of heterophils in the first week of life w24,23,25x. The pathogenic caecal species, E. colchici, which is the causative agent of acute coccidiosis
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in pheasants, is characterised by high morbidity and mortality rates, and produces a large number of oocysts in a white caseous exudate. According to Norton w3x the endogenous phase of E. colchici begins in the central portion of the small intestine with the formation of schizonts of the first generation 72᎐96 h p.i. The formation of second generation schizonts is described in the posterior portion of the small intestine. Norton w3x observed the infection of the caeca by the first schizonts of the third-generation as late as 96᎐120 h p.i. Oocyst production was observed 144 h after infection. In contrast, in our experiment first generation schizonts were found in the ileum and principally in the intestinal crypts of the caeca on day 1 p.i. w26x. Pellerdy ´ w2x reported that first generation schizonts released merozoites 60 h p.i. in the same portion of the intestine as in our trial. w2x found the beginning of gametogony Pellerdy ´ from the 106th h in the caeca, while the third shizogony cycle was still in progress, and the first oocysts were discharged 6 days after infection. Cell-mediated immunity plays a major role in the poultry’s resistance to coccidiosis. Complex interactions involving coccidian parasites with various sub-populations of intestinal lymphocytes are a characteristic feature of the gut immune responses to this intracellular microorganism w5x. After primary infection there were, in both the epithelium and the lamina propria, two distinct increases in the number of T-lymphocytes on days
Table 1 Absolute counts of leukocytes and lymphocytes in the peripheral blood of pheasant chicks infected by Eimeria colchici a Days after infection
1 2 3 4 5 6 14 Control 1᎐6 Control 14 a
Absolute numbers Ž1 = 109 ly1 . " S.D. Leukocytes
Lymphocytes
10.05" 2.72 19.58" 6.99U 19.77" 8.73U 20.13" 0.88UUU 13.14" 7.78 11.96" 2.46 27.55 " 5.06 10.22" 0.58 23.57" 2.85
8.58" 2.38 17.37" 5.86UU 17.12" 7.84U 18.53" 1.06UUU 11.12" 6.63 9.94" 2.24 23.43" 3.73 8.55" 1.16 21.55" 3.14
Significantly different from the days 1᎐6 controls: U P- 0.05; UU P- 0.01; UUU P- 0.001.
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Fig. 4. Proliferation of lymphocytes from the peripheral blood of pheasant chicks infected by E. colchici.
3᎐5 and then on day 11 after infection w8x. Our results demonstrate that the oral infection of pheasant chicks with coccidia results in lymphocytosis and leucocytosis at the beginning of the infection. Similar changes in the numbers of these cells have been observed in chickens and rats infected with coccidia w27x. The gradual decrease
of peripheral blood lymphocytes and leukocytes after day 4 may be due to the infiltration of lymphocytes into the intestinal mucosa at the site of infection w28,29x. The drop in the level of white blood cells after day 4 p.i. also coincides with the beginning of the sexual phase of the endogenous cycle of E. colchici in the colon of infected pheas-
Fig. 5. Index of metabolic activity of phagocytes from the peripheral blood of pheasant chicks infected by E. colchici.
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ants w26x. This closely coincides with the significant increase in the index of metabolic activity ŽIMA. of phagocytes, which was noted on day 3, 4 and 14 p.i. The increase of the IMA mainly in pheasant chicks of experimental group and the total number of leukocytes and lymphocytes on day 14 in both experimental and control pheasant chicks was possibly due the secondary bacterial infection of pheasant chicks in combination with age factors, because heterophil activities are agedependent in poultry with optimal levels at 14᎐21 days post-hatch with simultaneous development of cell-mediated immune responsiveness w24,23,25x. A significant increase in the proliferative activity of peripheral blood lymphocytes to E. colchici antigen on day 5 and 6 was noted with a maximum on day 14 p.i. Similar results were described by Lillehoj w30x in experimental coccidiosis of chickens. T-cell proliferative response to sporozoite antigens was detected on days 7᎐10 p.i. The peak T-cell proliferative response was observed 14 days p.i. and quickly waned after 21 day p.i. A direct relationship between resistance to infection E. tenella in chickens and lymphoproliferation of peripheral blood lymphocytes in response to parasite antigen was described w31x. In conclusion, these studies indicate that E. colchici infection of pheasant chicks resulted in a patent period lasting from day 7᎐11 p.i., with the largest production of oocysts occurring on days 8᎐9. The populations of peripheral blood heterophils and lymphocytes reflect the cellular immune response in a coccidial infection of pheasant chicks, which is characterised by leucocytosis at the beginning of coccidial infection, with increased phagocytosis of the heterophils and the proliferation response of lymphocytes to sporozoite antigens.
Acknowledgements This work was supported by a research grant from the Scientific Grant Agency of The ministry of Education of The Slovak Republic and Slovak Academy of Sciences ŽGrant 1r4254r97..
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